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Virus–Host Interaction Gets Curiouser and Curiouser. PART I: Phage P1vir Enhanced Development in an E International Journal of Molecular Sciences Article Virus–Host Interaction Gets Curiouser and Curiouser. PART I: Phage P1vir Enhanced Development in an E. coli DksA-Deficient Cell Grzegorz M. Cech 1,* , Anna Kloska 2 , Klaudyna Krause 1, Katarzyna Potrykus 1 , Michael Cashel 3 and Agnieszka Szalewska-Pałasz 1 1 Department of Bacterial Molecular Genetics, University of Gda´nsk,Wita Stwosza 59, 80-308 Gda´nsk,Poland; [email protected] (K.K.); [email protected] (K.P.); [email protected] (A.S.-P.) 2 Department of Medical Biology and Genetics, University of Gda´nsk,Wita Stwosza 59, 80-308 Gda´nsk,Poland; [email protected] 3 Intramural Program, Eunice Kennedy Shriver Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] * Correspondence: [email protected]; Tel.: +48-58-523-60-25 Abstract: Bacteriophage P1 is among the best described bacterial viruses used in molecular biology. Here, we report that deficiency in the host cell DksA protein, an E. coli global transcription regulator, improves P1 lytic development. Using genetic and microbiological approaches, we investigated several aspects of P1vir biology in an attempt to understand the basis of this phenomenon. We found several minor improvements in phage development in the dksA mutant host, including more efficient adsorption to bacterial cell and phage DNA replication. In addition, gene expression of the main Citation: Cech, G.M.; Kloska, A.; repressor of lysogeny C1, the late promoter activator Lpa, and lysozyme are downregulated in the Krause, K.; Potrykus, K.; Cashel, M.; dksA imm Szalewska-Pałasz, A. Virus–Host mutant. We also found nucleotide substitutions located in the phage immunity region I, Interaction Gets Curiouser and which may be responsible for permanent virulence of phage P1vir. We suggest that downregulation Curiouser. PART I: Phage P1vir of C1 may lead to a less effective repression of lysogeny maintaining genes and that P1vir may be Enhanced Development in an E. coli balancing between lysis and lysogeny, although finally it is able to enter the lytic pathway only. DksA-Deficient Cell. Int. J. Mol. Sci. The mentioned improvements, such as more efficient replication and more “gentle” cell lysis, while 2021, 22, 5890. https://doi.org/ considered minor individually, together may account for the phenomenon of a more efficient P1 10.3390/ijms22115890 phage development in a DksA-deficient host. Academic Editor: Giovanni Maga Keywords: P1vir bacteriophage; P1 phage; DksA; lytic development; host-virus interactions Received: 30 April 2021 Accepted: 26 May 2021 Published: 31 May 2021 1. Introduction λ Publisher’s Note: MDPI stays neutral Bacteriophage P1, along with phage and T4, is among the best described bacterial with regard to jurisdictional claims in viruses in molecular biology. The P1 features, as well as its life cycle, have been studied published maps and institutional affil- over many years [1], and its complete genome was published in 2004 [2]. The P1 phage iations. ability to conduct generalized transduction made it a valuable tool in molecular biology [3], used for genetic engineering and gene transfer, a role that even nowadays should not be underestimated [4]. Moreover, P1 served as a model to study various processes in molecular biology, such as DNA replication, recombination and multiple aspects of phage- host interactions, as widely summarized in [1]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. P1 can infect a variety of Gram-negative hosts, including many species from the This article is an open access article Enterobacteriaceae family [5], and its interactions with the host were mainly studied in distributed under the terms and the Escherichia coli model. Upon infection and entry into the host cell, P1 genomic DNA conditions of the Creative Commons undergoes circularization mediated by phage-encoded Cre-dependent recombination [6]. Attribution (CC BY) license (https:// At that time, as a temperate phage, P1 makes a choice between two different developmental creativecommons.org/licenses/by/ strategies, i.e., lysis versus lysogeny. As a lysogen, P1 genome exists in the host cell as a 4.0/). large extrachromosomal genetic element—a low copy number plasmid [7]. Int. J. Mol. Sci. 2021, 22, 5890. https://doi.org/10.3390/ijms22115890 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 5890 2 of 15 The lysis–lysogeny decision is mediated by an immunity system which includes three immunity regions: immC, immT and immI[8]. It needs to be emphasized that the P1vir phage, a virulent strain used in this study, was not constructed but isolated in the late 1960s and described as harboring unknown mutation(s) in the secondary repressor region—immI[9]. The choice of the phage life strategy is determined by a molecular interplay between products of genes expressed immediately upon infection. The major players encoded by the immC region are the main repressor protein, C1, and the C1 repressor inactivator protein, Coi [10,11]. C1 binds to the operator sequences present in the P1 genome and represses transcription from promoters located near these operators; this is necessary for lysogeny to occur. The Coi protein binds directly to C1, blocking its function and promoting lysis. Thus, the balance between these two regulators determines the choice between lysis and lysogeny. In addition, the Ant protein (Ant1-Ant2 dimer), encoded in the immI region, acts as a secondary anti-repressor which binds C1. Thus, for establishing lysogeny, expression of the ant genes has to be blocked, and this is achieved by C1-mediated transcription repression of an operon containing the c4, icd and ant1/2 genes. However, full repression of ant expression also requires the action of C4 antisense RNA [12]. The icd gene in this operon plays a role in negative regulation of ant expression. Moreover, Icd inhibits host cell division which provides time needed for either establishing lysogeny or entering into the lytic pathway [13]. Yet another player in this complex regulation is the Lxc corepressor encoded by the immT region. Lxc enhances the ability of C1 to bind to the mentioned P1 operators [14]. Once lysogeny is established, the P1 genome replicates similarly to plasmid DNA. This plasmid has two replication origins, oriR and oriL, and exhibits a complex regulation of replication, partition and addiction [1]. Replication starts at oriR and it is initiated with P1 encoded RepA protein. The second origin, oriL, is involved during lytic development. Replication from both origins proceeds via the theta model, however later in the lytic development it switches to the sigma model, which enables phage heads’ packing [15]. The lytic pathway can be chosen upon infection, but changing environmental condi- tions may also provoke P1 lysogen to enter the lytic pathway. In that case, many genes formerly repressed by C1 undergo expression, including early genes whose products are responsible for initiation of the replication process and its control. Lytic replication is initi- ated at oriL by the RepL protein. The repL gene is co-transcribed with kilA which encodes a protein that inhibits cell division during lytic phage development. Other early proteins are involved in regulation of gene expression, recombination, and methylation [1]. The late genes, including those whose products are involved in phage morphogenesis, packaging of virus particles and cell lysis, are typically expressed from specific promoter sequences whose transcription requires a phage-encoded regulator, the Lpa protein, defined as an RNA polymerase binding factor [16]. Assembled and accumulated phage particles are released from the host cell due to the action of phage-encoded lysozyme, whose function is controlled by a system of holin and antiholin proteins (LydA and LydB) [17]. In their development, bacteriophages rely on and are affected by multiple host regu- latory processes and factors. DksA was identified as a regulatory protein affecting many processes, e.g., heat stress response [18], and its role as a transcription regulator involved in the stringent response [19,20] placed DksA among proteins intensively studied by many research groups. Its function in the regulation of various processes has been reported, including DNA repair, quorum sensing, and virulence [21–23]. Thus, DksA deficiency affects numerous cellular processes causing pleiotropic effects. We discovered that a host cell deficiency in DksA affects bacteriophage P1 life cycle leading to its improved lytic development; we attempt to understand the basis of this effect by using genetic and microbiological approaches. As the phage development is a multistep process, many factors including host features, the process of adsorption, the choice of the life strategy, the phage gene expression and virion forming, as well as the host cell lysis Int. J. Mol. Sci. 2021, 22, 5890 3 of 15 will affect the outcome of phage infection shown as a phage progeny number. Thus, the observation of the changes in the lytic development efficiency of a given phage may have an explanation at one of these numerous steps. Interestingly, the lack of DksA in E. coli cells was also reported to affect T4 phage development, resulting in increased plaque size and a more productive infection [24], however, the mechanism involved was quite different than what we report here for phage P1vir. Here, we present the first part of our scientific story which is getting more and more interesting or “curiouser and curiouser”, bringing to mind “Alice’s Adventures in Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 17 Wonderland” by Lewis Carroll. We would like to encourage the reader to follow this story in our accompanying paper: Virus–Host Interaction Gets Curiouser and Curiouser. PART II: Functional Transcriptomics of the E. coli DksA-Deficient Cell upon Phage P1vir Infection. 2. Results and Discussion 2. Results and Discussion 2.1.
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